Prev. Nutr. Food Sci. 2013;18(1):11-17 http://dx.doi.org/10.3746/pnf.2013.18.1.011 pISSN 2287-1098ㆍeISSN 2287-8602

Protective Effect of Padina arborescens Extract against High Glucose-induced Oxidative Damage in Human Umbilical Vein Endothelial Cells Mi Hwa Park and Ji Sook Han Department of Food Science and Nutrition, Pusan National University, Busan 609-735, Korea

ABSTRACT: Dysfunction of endothelial cells is considered a major cause of vascular complications in diabetes. In the present study, we investigated the protective effect of Padina arborescens extract against high glucose-induced oxidative damage in human umbilical vein endothelial cells (HUVECs). High-concentration of glucose (30 mM) treatment induced cytotoxicity whereas Padina arborescens extract protected the cells from high glucose-induced damage and significantly restored cell viability. In addition, lipid peroxidation, intracellular reactive oxygen species (ROS), and nitric oxide (NO) levels induced by high glucose treatment were effectively inhibited by treatment of Padina arborescens extract in a dose-dependent manner. High glucose treatment also induced the overexpressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and NF-κB proteins in HUVECs, but Padina arborescens extract treatment reduced the overexpressions of these proteins. These findings indicate the potential benefits of Padina arborescens extract as a valuable source in reducing the oxidative damage induced by high glucose. Keywords: Padina arborescens, diabetes, oxidative damage, high glucose, HUVECs

INTRODUCTION Diabetes is a common metabolic disease characterized by abnormally high plasma glucose levels, leading to major complications, such as diabetic neuropathy, retinopathy, and cardiovascular diseases (1). Several recent studies have demonstrated that hyperglycemia can cause glucose to undergo autooxidation to generate intermediates that lead to the formation of ROS, nitric oxide (NO), peroxynitrite (ONOO−), and advanced glycation end products (AGE), which cause various complications of diabetes (2). Prolonged hyperglycemia is the major factor in the etiology of atherogenic pathogenesis in diabetes, which causes 80% of total mortality in diabetic patients. One diabetic vascular complication involves endothelial dysfunction characterized by impaired endothelium-dependent vasomotor responses. Previous studies have shown that hyperglycemia induces endothelial dysfunction, possibly due to oxidative stress (3,4). Accordingly, a reduction in cellular antioxidant reserves is responsible for triggering diabetic vascular complications (5,6). Therefore, antioxidants can prevent pathological damage caused by hyperglycemia-induced oxidative stress

associated with diabetes (7). Marine algae have demonstrated free radical scavenging activities, and thus may help slow aging and prevent some chronic diseases. In particular, brown algae display a variety of biological activities, including antioxidant (8), anti-inflammatory (9), anti-coagulant (10) and anti-hyperlipidemic properties (11). Padina arborescens, a type of brown alga popular in Korea and Japan as a food ingredient and marine herb, contains biologically active compounds such as bromophenols (12). However, in high glucose, the effect of Padina arborescens on oxidative damage of HUVECs is unclear. In the present study, we investigated the protective effects of Padina arborescens extract (PAE) against high glucose-induced oxidative damage using human umbilical vein endothelial cells (HUVECs).

MATERIALS AND METHODS Materials The brown alga, Padina arborescens (Phylum Ochrophyta, Class Phaeophyceae, Order Dictyotales, Family Dictyota-

Received: November 19, 2012; Accepted: February 14, 2013 Correspondence to: Ji Sook Han, Tel: +82-51-510-2836, E-mail: [email protected] Copyright © 2013 by The Korean Society of Food Science and Nutrition. All rights Reserved. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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ceae) was collected along the coast of Jeju Island, Korea. The sample was washed three times with tap water to remove the salt, epiphytes, and sand attached to the surface, then carefully rinsed with fresh water and maintained in a medical refrigerator at −20oC. Thereafter, the frozen samples were lyophilized and homogenized with a grinder prior to extraction. Padina arborescens was extracted with ten volumes of 80% methanol for 12 h three times at room temperature. The filtrate was then o evaporated at 40 C to obtain the methanol extract. The Padina arborescens extract (PAE) was thoroughly dried for complete removal of solvent and stored in a deep freezer o (Nihon Freezer Co., Tokyo, Japan) (−80 C). Cell culture Human umbilical vein endothelial cells (HUVECs) and the endothelial cell basal medium-2 (EBM-2) bullet kit were purchased from Clonetics Inc. (San Diego, CA, USA). Cells were cultured in EGM-2 containing 2% fetal bovine serum (FBS; GIBCO Inc., Grand Island, NY, USA), at 37oC in a humidified atmosphere containing 5% CO2 according to the supplier’s recommendations, and used between passages 3 and 6. Assay of neutral red cell viability Cell viability was assessed by measuring the uptake of the supravital dye neutral red (13). Cells (4×104 cells/ well) cultured in 24-well plates were pre-incubated with glucose (5.5 and 30 mM) in humidified atmosphere containing 5% CO2 at 37oC for 48 h. After 48 h of incubation, the cells were treated with various concentrations (25, 50, and 100 μg/mL) of PAE and further incubated for 20 h. Thereafter, the medium was carefully removed from each well, and replaced with 0.5 mL of fresh medium containing 1.14 mmol/L neutral red. After 3 h of incubation, the medium was removed and the cells were washed twice with phosphate buffered saline (PBS, pH 7.4). The incorporated neutral red was released from the cells by incubation in the presence of 1 mL of cell lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 5 mmol/L dithiothreitol (DTT), and Triton X-100 (1%, v/v)] containing acetic acid (1%, v/v) and ethanol (50%, v/v) at room temperature for 15 min. To measure the dye taken up, the cell lysis products were centrifuged and absorbance of the supernatant was measured spectrophotometrically at 540 nm (Bio-Rad Laboratories Inc., Hercules, CA, USA). Assay of lipid peroxidation Lipid peroxidation, which was caused by influence of ROS generated with a high glucose-induced oxidative damage in the cells, was measured by thiobarbituric acid reactive substances (TBARS) production. Cells (4×104 cells/well) were seeded in 24-well plates and pre-incu-

bated with glucose (5.5 and 30 mM) in humidified atmosphere containing 5% CO2 at 37oC for 48 h. After 48 h of incubation, the cells were treated with various concentrations (25, 50, and 100 μg/mL) of PAE and further incubated for 20 h. A 200 μL sample of each medium supernatant was mixed with 400 μL of TBARS solution o and then boiled at 95 C for 20 min. The absorbance at 532 nm was measured and TBARS concentrations were extrapolated from the 1,1,3,3-tetraethoxypropane serial dilution standard curve. TBARS values were then expressed as equivalent nmoles of malondialdehyde (MDA) (14). Assay of intracellular ROS levels Intracellular ROS levels were measured by the 2’,7’-dichlorofluorescein diacetate (DCF-DA) assay (15). DCFDA can be deacetylated in cells by reacting quantitatively with intracellular radicals to convert into its fluorescent product, DCF, which is retained within the cells. Therefore, DCF-DA was used to evaluate the generation of ROS in oxidative damage. Cells (2×104 cells/well) were seeded in 96-well plates and pre-incubated with glucose (5.5 and 30 mM) in humidified atmosphere containing 5% CO2 at 37oC for 48 h. After 48 h of incubation, the cells were treated with various concentrations (25, 50, and 100 μg/mL) of PAE and further incubated for 20 h. Thereafter, the medium was removed and the cells were washed twice with phosphate buffered saline (PBS, pH 7.4) and incubated with 100 μM DCF-DA for 90 min at room temperature. Fluorescence was measured using a fluorescence plate reader (BMG LABTECH GmbH, Offenburg, Germany). Assay of nitric oxide (NO) levels The amount of nitrite accumulation, the end product of NO generation, was assessed by the Griess reaction 4 (16). Cells (2×10 cells/well) were seeded in 96-well plates and pre-incubated with glucose (5.5 and 30 mM) in humidified atmosphere containing 5% CO2 at 37oC for 48 h. After 48 h of incubation, the cells were treated with various concentrations (25, 50, and 100 μg/mL) of PAE and further incubated for 20 h. Thereafter, each 50 μL of culture supernatant was mixed with an equal volume of Griess reagent [0.1% N-(1-naphthyl)-ethylenediamine, 1% sulfanilamide in 5% phosphoric acid] and incubated at room temperature for 10 min. The absorbance at 550 nm was measured in a microplate absorbance reader and a series of known concentrations of sodium nitrite was used as the standard curve. Total and nuclear protein extracts Cells were homogenized with ice-cold lysis buffer containing 250 mM NaCl, 25 mM Tris-HCl (pH 7.5), 1% v/v NP-40, 1 mM DTT, 1 mM PMSF, and protein inhibitor

Padina arborescens Extract and Glucose Oxidative Damage

cocktail (10 μg/mL aprotinin, 1 μg/mL leupeptin). The cells were then centrifuged at 20,000×g for 15 min at 4oC. The supernatants were used as total protein extracts (17). For nuclear protein extracts, cells were homogenized with ice-cold lysis buffer containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 15 mM CaCl2, 1.5 M sucrose, 1 mM DTT, and protease inhibitor cocktail (10 μg/mL aprotinin, 1 μg/mL leupeptin). Then, the cells were centrifuged at 11,000×g for 20 min at 4oC. The supernatants were resuspended with extraction buffer containing 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, 25% (v/v) glycerol, 10 mM DTT, and protease inhibitor cocktail (10 μg/mL aprotinin, 1 μg/mL leupeptin). The samples were shaken gently for 30 min and centrifuged at 21,000×g for 5 min at 4oC. The pellets were used as nuclear protein extracts. The total and nuclear protein contents were determined by the Bio-Rad protein kit (Bio-Rad Laboratories Inc.) with BSA as the standard. Immunoblotting iNOS and COX-2 expressions and NF-κB p65 DNAbinding activity were determined by western blot analysis (17). Total protein (20 μg) for iNOS and COX-2 protein levels and nuclear protein (20 μg) for NF-κB were electrophoresed through 10% sodium dodecyl sulfatepolyacrylamide gel (SDS-PAGE). Separated proteins were transferred electrophoretically to a pure nitrocellulose membrane, blocked with 5% skim milk solution for 1 h, and then incubated with primary antibodies (Abcam, o Cambridge, UK; 1:1,000) overnight at 4 C. After washing, the blots were incubated with goat anti-rabbit or goat

Fig. 1. Effect of PAE on high glucose-induced-oxidative damage of HUVECs. HUVECs were preincubated with normal glucose (5.5 mM) and high glucose (30 mM) for 48 h. Thereafter, HUVECs were treated with various concentrations (25, 50, and 100 μg/mL) of PAE and further incubated for 20 h. After an incubation of 20 h, cell viability was determined by neutral red assay. Each value is expressed as mean±SD in triplicate experia-e ments. Values with different alphabets are significantly different at p

Protective Effect of Padina arborescens Extract against High Glucose-induced Oxidative Damage in Human Umbilical Vein Endothelial Cells.

Dysfunction of endothelial cells is considered a major cause of vascular complications in diabetes. In the present study, we investigated the protecti...
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